Micro and nanotechnologies: The little formulations that could

Abstract The first publication of micro‐ and nanotechnology in medicine was in 1798 with the use of the Cowpox virus by Edward Jenner as an attenuated vaccine against Smallpox. Since then, there has been an explosion of micro‐ and nanotechnologies for medical applications. The breadth of these micro‐ and nanotechnologies is discussed in this piece, presenting the date of their first report and their latest progression (e.g., clinical trials, FDA approval). This includes successes such as the recent severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines from Pfizer, Moderna, and Janssen (Johnson & Johnson) as well as the most popular nanoparticle therapy, liposomal Doxil. However, the enormity of the success of these platforms has not been without challenges. For example, we discuss why the production of Doxil was halted for several years, and the bankruptcy of BIND therapeutics, which relied on a nanoparticle drug carrier. Overall, the field of micro‐ and nanotechnology has advanced beyond these challenges and continues advancing new and novel platforms that have transformed therapies, vaccines, and imaging. In this review, a wide range of biomedical micro‐ and nanotechnology is discussed to serve as a primer to the field and provide an accessible summary of clinically relevant micro‐ and nanotechnology platforms.


| DEFINING MICRO-AND NANOTECHNOLOGY
Although the concept of nanotechnology was first introduced by Richard Feynman in 1959 1,2 and the term defined by Norio Taniguchi in 1974, 2,3 organic and inorganic materials that can be characterized as nanotechnology already existed. This is because the definition is based on size-they are simply technological products that have nanometer (nm) dimensions, and although they are applied in a variety of scientific and technical fields, this review will be highlighting nanotechnology through a biomedical lens. By National Institutes of Health (NIH) standards, nanotechnology ranges between 1 and 100 nm in size. 4 By this strict definition, there are organic components common to the biological world ( Figure 1) that fit within the scope of nanotechnology, such as DNA, protein, and viruses. These nano-scale biological components have a longstanding history of clinical benefit. One very impactful example was Edward Jenner's development of Cowpox virus as a Smallpox vaccine in 1798 5,6 ( Figure 2). This vaccine alone saved countless lives and facilitated the global eradication of the deadly disease Smallpox. 7 The use of nanotechnology to eradicate a deadly disease underscores the impact of nanotechnologies and their influence in our world, even before they were given a definition.
Beyond nanotechnology and its historical definition, the range of these biomedical platforms has been tremendous ( Figure 2; Table 1). Rebeca T. Stiepel and Eliza Duggan contributed equally to this work.
This review focuses on why biomedical technologies in this size range are unique, as well as the highs and lows of these technologies in medical applications. To give the breadth of successes of these platforms, a timeline of discovery of the most referenced micro-and nanotechnologies is illustrated and presented with a short description and information related to their discovery, clinical trials, and F I G U R E 1 Scale of biological world. DNA, deoxyribonucleic acid; nm, nanometer F I G U R E 2 Timeline of first reports of micro and nanotechnology used for therapeutic, vaccine, and imaging applications. CAR T cells, chimeric antigen receptor T cells; NP, nanoparticle; PEG, polyethylene glycol; PEGylation/PEGylated, PEG covalently bound; PRINT, particle replication in nonwetting templates T A B L E 1 First reports for micro-and nanotechnologies In addition to having a higher surface area to volume ratio, the smaller scale of micro-and nanomaterials allows for specific interac-

| Polymer based
There are several micro and nanotechnologies comprised mostly of polymers ( Table 2). In 1960, an early report on polymeric technologies focused on hydrogels. 44 Hydrogels are made of crosslinkedhydrophilic polymers that traditionally swell in the presence of water, making them ideal for use in soft-contact lenses, for which they were first approved in 1971. 45 Hydrogels are also used as drug delivery systems to achieve a controlled release of therapeutic cargo over time.
Additional drug delivery platforms are often comprised of polymeric particles, which are often created by emulsion (e.g., homogenization, sonication) or spray (e.g., electrospray, spray drying) methods. Both types of methods can successfully form polymeric particles encapsulating a variety of cargos. Although, high speed shearing associated with emulsion methods can negatively affect protein antigens, 142 and there are some limitations of solvent selection with spray methods. 143,144 The best fabrication method can vary by cargo and application of the particle system. These systems typically employ a hydrophobic polymer, which does not need to be cross- Polymer-based micro and nano-structures can also be fabricated by micromolding, a technique wherein a master structure is created through microfabrication, usually from silica. A mold is then made from the master, typically out of polydimethylsiloxane (PDMS).
Another polymer solution is poured into the mold and left to harden.
Once removed, the hardened polymer is in the shape of the master. 145 Two examples of micromolding are microneedles and particle replication in nonwetting templates (PRINT) particles. Microneedles T A B L E 2 Estimated percent of atoms on the surface of nanometer to meter scaled materials assuming a sphere packing density of $74% were originally reported in 1998 125 and comprised of inorganic materials. 145 Eventually, the inorganic materials were used as masters for the fabrication of polymeric microneedles. 146 In 2001, the first clinical trial for microneedles was conducted to evaluate pain upon application, validating the platform as a painless transdermal drug delivery system. 126 In the case of PRINT particles, micromolding techniques were modified to use a highly fluoridated polymer to create a mold that resists wetting. With this modification, nano and micro-scaled particles have been made. These PRINT particles were first reported in 2005 128 and advanced to clinical trials as an influenza vaccine adjuvant in 2010. 129 The ability to tune polymer properties through monomer selection and copolymerization makes this material class highly tunable for biomedical applications. One example of this is the generation of amphiphilic macromolecules that exhibit both hydrophilic and lipophilic properties by forming a copolymer of a hydrophilic monomer/ polymer (e.g., polyethylene glycol (PEG)) and a lipophilic monomer/  ticles with dense PEG chains in a long "brush" morphology are less likely to be taken up by cells than particles with less dense PEG chains in a shorter "mushroom" morphology. 153 First identified in 1977, 85 PEGylated adenosine deaminase (Adagen) was the first PEGylated therapy approved in 1990, 86 though it was later discontinued from the market due to insufficient supply of bovine adenosine deaminase. 154 Due to the ubiquity of PEGylation applied to micro-and nanotechnology, further discussion on PEG applied to drug delivery platforms can be found in the following sections: "Success can Come in Small Packages" and "Challenges in the Field." Although PEG has been used to functionalize many drug carriers, functionalization via the addition of a targeting group is a significant feature in micro-and nanotechnology. The addition of targeting most often refers to active targeting ( Figure 6), which can allow enrichment of a drug or carrier in a site of interest, such as a tumor. By enriching the drug or carrier in an area of interest, the off-target effects should be reduced while the amount of desired cargo at the region of interest is increased (e.g., tumor site). In 1992, 117 the first study was reported wherein a PEGylated liposome was functionalized at the end of the PEG chain with a targeting ligand to achieve longer circulation times and increase delivery to necrotic sites after myocardial infarction. This work continues to be preclinical, primarily for the delivery of therapeutic agents for cancer.

| Nonlipid biologics
With the inclusion of microtechnologies, the scale ( Figure 1) moves beyond 100 nm to include nonviral microbes (e.g., bacteria, yeast, algae) and mammalian cells. Nonviral microbe-based therapies were reported as early back as 1813 when the bacteria Clostridium perfringens was used to slow the growth of a tumor in a cancer patient. 9 More recently bacteria as a platform has been used clinically for fecal microbiota transplants (FMTs) to treat Clostridium difficile infection, 160 and FMTs are being evaluated for other conditions associated with microbiome dysbiosis, including but not limited to inflammatory bowel disease. 161  F I G U R E 5 Graphic highlighting the properties that PEGylation can impart on micro-and nanotechnology-based formulations. 150,151 F I G U R E 6 Graphic contrasting passive and active targeting with two examples. Passive targeting is exemplified by using size to exclude uptake in most cells, for preferential uptake in phagocytic cells. Active targeting is illustrated by using an antibody fragment against a receptor (EGFR) that is upregulated on cancer cells. 6,155 These mammalian cell-based therapies illustrate a more tailored approach that can require patient samples (e.g., blood, tumor) and perhaps genetic sequencing to develop a more personalized treatment.
For example, in the generation of CAR T cells, 162

| SUCCESS CAN COME IN SMALL PACKAGES
Perhaps foreshadowed by Jenner's Smallpox vaccine which prevented substantial morbidity and mortality during a deadly pandemic, the most significant success of nanotechnology in the 21st century was the rapid production of lipid nanoparticles (LNPs) and adenoviruses for vaccination against SARS-CoV2, the virus that causes COVID-19. 156 Using LNPs with ionizable lipids, 163  approved. Just as a copolymer has the properties of each of its separate monomers, the addition of PEG to a therapy can make a hydrophobic compound more water soluble. PEG is so significantly waterloving that it is thermodynamically unfavorable for water to disassociate from the polymer 170 ( Figure 5). Because water would need to be displaced for protein adhesion, and protein adhesion facilitates cell attachment, PEG imparts resistance to both protein and cell adhesion. Long acting cabotegravir and rilpivirine have been developed as intramuscular injected nanocrystals for sustained delivery of drug lasting for a month or more. 183 Although oral delivery is thought to be paramount in drug delivery, the rate of adherence to oral ART is approximately 70% independent of assessment method. This is concerning considering that virologic failure (the inability to maintain suppression of viral replication) and drug resistance significantly increase when adherence rates fall below 90%-95%. 184

| CHALLENGES IN THE FIELD
Since micro-and nanotechnology platforms have unique interactions with the biological world, a logical application would be to use them to better target cells and tissues of interest. These materials can be used to passively target areas of the body ( Figure 6). For instance, micron sized particles can passively target phagocytic immune cells over nonphagocytic cells which typically only internalize material less than 200 nm ( Figure 6). 6 Most notably in drug delivery research, nanomaterials have been noted to accumulate in tumors and at sites of inflammation due to the enhanced permeability and retention (EPR) effect ( Figure 7). 186 The EPR effect occurs in solid tumors when the tumor reaches 1-2 centimeters in size, and can no longer feed all of its cells through diffusion from peripheral capillaries. 187 The formation of capillaries by the cancer becomes haphazard and results in leaky junctions between endothelial cells and pores as large as 200 nm. 185 However, there is controversy regarding actual accumulation of NPs in tumors due to EPR, 188 despite evidence that the drug fraction in the tumor compared to the blood is improved with delivery via NPs. 189 Some of this controversy can be attributed to observed differences between cancer in mouse models and humans including increased vascular density in several mouse models compared to human tumors. 190 Also there is concern of poor NP permeation into tumors is due to the high intratumoral pressure upon more advanced tumor development, which can be the result of poor lymphatic drainage in the tumor and eventual buildup of fluids in an acidic and hypoxic environment. 191 With high intratumoral pressure, the penetration of NPs and even chemotherapeutic agents into the tumor can become limited. Concern regarding EPR-mediated passive targeting of nanomaterials has led to significant criticism of these technologies for cancer chemotherapy. 192 Therefore, scientists who develop, characterize, and apply these materials clinically have a responsibility to be rigorous and transparent in their analysis so as to better mitigate con- were bought by Pfizer. 195,196 While this represents a high-profile setback for the field of nanomedicine, without difficulty the field cannot advance to the many successes that have come about today. Indeed, critical evaluation of the EPR effect and the use of targeted nanotherapeutics have prompted new insights into fundamental aspects of particle transport into tumors, 197,198 and more broadly there is consistent growth in understanding the use of NPs as therapeutics. This understanding is highlighted in the wealth of nanotechnology that has made it to clinical trials and has received FDA approval (Table 1). 32,199 As Other drawbacks of these formulations are in line with those observed for many classes of therapies, including increased formulation production cost, adverse toxicity, and potential off target immune responses. 212,213 Although Ambisome has been a transformative technology for leishmaniasis, the cost limits the ability of the treatment to be widely administered compared to conventional therapies that are less expensive. For many families in endemic regions, the cost of Ambisome treatment is often greater than their yearly income. 214 One way that micro-and nanotechnologies can reduce cost is through reduction in supportive care 171 for toxic side-effects noted with conventional formulations; however, the addition of polymers, lipids, and other materials can create additional toxicity concerns. One example is hand-foot syndrome where chemotherapy from the formulation leaks into the hands and feet causing redness, swelling, and pain. 215 Hand-foot syndrome is observed with Doxil treatment because of the long circulation times of the PEGylated liposome. 216

ACKNOWLEDGMENTS
The authors would like to thank Dr. Liubov Lifshits for help with an illustration. Illustrations were generated by Biorender. Funding was provided in part by NIH NIAID R01AI147497.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.